PROJECT SUMMARY Radiation nephropathy (RN) is less common than chemotherapy-induced nephrotoxicity but still represents a serious late complication after radiation therapies for cancer. RN is irreversible and no effective clinical treatments exist to prevent RN or ameliorate radiation-associated kidney injury. Podocyte loss, tubular atrophy and endothelial damage have been linked with RN, but the molecular mechanisms governing RN are not known. We discovered that the enzyme sphingomyelin-phosphodiesterase-acid-like-3b (SMPDL3b) is an important regulator of radiation damage in renal podocytes after single dose (SD) radiotherapy (RT). Radiation damage reduced SMPDL3b expression triggering the cellular relocation of ezrin and a morphological change that altered podocyte functionality. Treatment with rituximab, which we demonstrated to bind SMPDL3b and to protect podocyte morphology, reduced SD RT induced RN in C57BL/6 mice but not in our newly-developed conditional podocyte-specific SMPDL3b knock-out mice. Based on these data we hypothesize that sphingolipids play a vital role in radiation-induced podocytopathy which governs RN. The objective is to investigate the mechanistic role of SMPDL3b in renal injury after fractionated low-dose radiotherapy (F-RT) with concurrent cisplatin (CDDP) as this represents a standard of care for many solid cancers. Our long-term goal is to discover a molecular-based protective or mitigating strategy for RN, and potentially chemotherapy-induced nephrotoxicity. We will test our hypothesis with the following three specific aims using a combined in vivo-in vitro approach: Aim 1: To determine if SMPDL3b regulates severity and latency of RT-associated kidney injury and functional RN after clinically-relevant F-RT, CDDP and concurrent F-RT+CDDP. This aim will also explore the role of SMPDL3b in tissue tolerance for RT retreatment injury, using C57BL/6 mice and our unique SMPDL3b-knockout and SMPDL3b-inducible mouse models. Aim 2: To determine the mechanism by which podocyte expression of SMPDL3b affects RT-mediated podocyte and glomerular endothelial cell (GEC) injury. We hypothesize that SMPDL3b affects RT induced compartmentalization of podocyte ezrin and affects GECs via altered endothelin-1 (EDN1) and END1 receptor type A (EDNRA) cross talk. GEC survival after RT will be studied by co-culturing GECs with podocytes lacking or expressing SMPDL3b. Aim 3: To determine if targeting sphingolipids prevents RN. We will investigate if protection of SMPDL3b or S1P will avert long-term functional renal injury in C57BL/6 mice after F-RT, CDDP and F-RT+CDDP. Mechanisms will be confirmed using our unique SMPDL3b-knockout and SMPDL3b-inducible mouse models. The findings from these studies will be significant because they offer the potential for molecular-targeted mitigation for RN, and radiation-associated kidney injury, after RT and combined modality injury.